Stable no-delivering compounds

ABSTRACT

Disclosed are novel NO-releasing compounds which comprise a stabilized S-nitrosyl group and a free alcohol or a free thiol group. Also disclosed is a method of preparing the NO-releasing compounds. The method comprises reacting a polythiol or a thioalcohol with a nitrosylating agent. Also disclosed are medical devices coated with the disclosed compounds, methods of delivering NO to treatments sites in a subject by utilizing the medical devices and methods of sterilizing surfaces.

RELATED APPLICATIONS

This application is a continuation of application Ser. No. 10/217,580,filed Aug. 12, 2002, now U.S. Pat. No. 6,583,311, which is acontinuation of application Ser. No. 10/026,851, now U.S. Pat. No.6,488,951, filed Dec. 18, 2001, which is a continuation of applicationSer. No. 09/725,603, now U.S. Pat. No. 6,359,167, filed Nov. 29, 2000which is a continuation of U.S. application Ser. No. 09/103,227, nowU.S. Pat. No. 6,207,855, filed Jun. 23, 1998. The entire teachings ofthe above applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Nitric oxide (referred to herein as “NO”) has many uses, including as amedicinal agent. For example, NO has been shown to inhibit smooth muscleproliferation, thrombus formation, platelet aggregation, and smoothmuscle contraction. NO can also be used as a bacteriocidal orbacteriostatic agent to sterilize the surfaces of, for example, medicaldevices. However, the half-life for NO release of most of the knowncompounds which release NO is less than twelve hours. Thus, most knownNO-releasing compounds are too unstable to be useful commercially. Thefull commercial potential of NO is unlikely to be realized until morestable NO-releasing compounds are developed.

SUMMARY OF THE INVENTION

It has now been found that an S-nitrosyl group (an “—S—NO” group) in acompound generally is stabilized when the compound also has a free thiolgroup or a free alcohol group. For example, the half-life for NO-releasefrom thiol or alcohol bearing S-nitrosylated compounds is generallygreater than about two hundred hours when nitrosylated with betweenabout 0.5 to about 0.7 equivalents ofS-nitroso-N-acetyl-D,L-penicillamine (SNAP) (Example 2). In contrast,the half-life for NO release from most S-nitrosylated compounds withoutfree thiols or alcohols is typically less than twelve hours. Based onthese results, novel compounds with stabilized S-nitrosylated groups andmethods of preparing these compounds are disclosed. Also disclosed aremedical devices coated with the disclosed compounds, methods ofdelivering NO to treatments sites in a subject by utilizing the medicaldevices and methods of sterilizing surfaces.

One embodiment of the present invention is an NO-releasing compoundcomprising an S-nitrosyl group and a free alcohol or a free thiol group.The S-nitrosyl group is stabilized with the alcohol or thiol group.

Another embodiment of the present invention is a compound prepared byreacting a polythiol or a thioalcohol with a nitrosylating agent.Preferably, from about 0.5 to about 0.7 equivalents of nitrosylatingagent for each free thiol and each free alcohol group in the polythiolor thioalcohol is used. A “polythiol” is a compound with at least twofree thiol groups. A “thioalcohol” is a compound with at least one freealcohol and at least one free thiol group.

Yet another embodiment of the present invention is a method of preparingan NO-releasing compound comprising at least one S-nitrosyl group and atleast one free alcohol or free thiol group, wherein the S-nitrosyl groupis stabilized with the alcohol or thiol group. The method comprisesreacting a polythiol or a thioalcohol with a nitrosylating agent.Preferably, from about 0.5 to about 0.7 equivalents of nitrosylatingagent for each free thiol and each free alcohol group in the polythiolor thioalcohol are used.

Another embodiment of the present invention is an article which iscapable of releasing NO. The article contains (e.g., incorporates or iscoated with) at least one of the compounds of the present invention. Thearticle can be a device for which a useful result can be achieved by NOrelease, including a medical device suitable for implantation at atreatment site in a subject. The medical device can deliver nitric oxideto the treatment site in the subject after implantation. In anotherexample, the article is, for example, a tube or catheter for contactinga bodily fluid of a subject.

Another embodiment of the present invention is a method of deliveringnitric oxide to a treatment site in a subject. A medical device whichcontains one or more of the compounds of the present invention isimplanted into the subject at the treatment site.

Another embodiment of the present invention is a method of deliveringnitric oxide to a bodily fluid of a subject. The method comprisescontacting the bodily fluid with an article, for example a tube orcatheter, which contains at least one of the compounds of the presentinvention.

Yet another embodiment of the present invention is a method ofinhibiting the growth of bacteria on surfaces. The method comprises thestep of contacting the surface with an effective amount of a compound ofthe present invention.

The compounds of the present invention have longer half-lives forNO-release than most known NO-releasing compounds. Thus, the compoundsof the present invention can be remain at internal treatment sites forlonger durations when used as a coating for implantable medical devicesand can be stored for longer periods of time than most knownNO-releasing compounds. They can also be used as bacteriostatic agents.

DETAILED DESCRIPTION OF THE INVENTION

The NO-releasing compounds of the present invention are small organicmolecules. Thus, the compounds of the present invention are comprisedprimarily of carbon and hydrogen, but can also include othernon-metallic elements such as sulfur, oxygen, nitrogen and/or halogens.The compounds of the present invention can contain functional groupswhich do not substantially increase the rate of NO release, for example,double the rate of release compared with the corresponding compoundwithout the functional group. Examples of suitable functional groupsinclude alcohols, thiols, amides, thioamides, carboxylic acids,aldehydes, ketones, halogens, double bonds, triples bonds and arylgroups (e.g, phenyl, naphthyl, furanyl, thienyl and the like).

As used herein, the term “small organic molecule” excludesmacromolecules such as a polypeptides, proteins, or S-nitrosylatedpolysaccharides or polymers, such as those disclosed in co-pending U.S.Ser. No. 08/691,862. The entire teachings of U.S. Ser. No. 08/691,862are incorporated herein by reference. The invention also excludesS-nitrosylated derivatives of the compounds represented by StructuralFormula (I)–(VII):

Each Ra is —H or methyl and is independently chosen.

As used herein, a “compound with a stabilized S-nitrosyl group”comprises, along with the S-nitrosyl group, a free thiol group or freealcohol group and has a half-life for NO release which is significantlygreater than for the corresponding compound with no free thiol oralcohol group (e.g., at least two times greater, and often about tentimes greater) when the same nitrosylating agent has been used toprepare both compounds. For example, the half-life for NO release for6-S-nitrosyl-hexane-1-thiol is about 1800 hours when prepared with SNAP(Example 2), whereas the half-life for NO release for1-S-nitrosyl-hexane is just over 200 hours when prepared with SNAP.Thus, 6-S-nitrosyl-hexane-1-thiol has a stabilized S-nitrosyl group.Generally, a compound with a stabilized S-nitrosyl group has a half lifefor NO release greater than about two hundred hours when nitrosylatedwith between about 0.5 to about 0.7 equivalents of SNAP, and oftengreater than about one thousand hours.

At least one —S-nitrosyl group in the disclosed compounds is stabilizedby the interaction between a free thiol or a free alcohol group and the—S-nitrosyl group. Although Applicants do not wish to be bound by anyparticular mechanism, this stabilization is consistent with the—S-nitrosyl group and free thiol (or alcohol) existing in equilibriumwith a cyclic structure, as shown below in Structural Formula (VIII):

—Y— is —O— or —S—. The “dashed” lines in Structural Formula (VIII)represent a stabilizing interaction, for example, a partial bond between—Y— and the sulfur atom and between —Y— and the nitrogen atom. Astabilizing interaction can be formed, for example, when a free thiol oralcohol is located within three covalent bonds of (alpha to) anS-nitrosyl group. In another example, a stabilizing interaction can beformed when a free thiol or alcohol can be brought within about one toone and a half bond lengths of an S-nitrosyl group by energeticallyaccessible conformational rotations of covalent bonds within themolecule.

Based on the discussion in the previous paragraph, the compounds of thepresent invention can also be described as comprising one or more of thecyclic structure shown in Structural Formula (VIII). The compounds ofthe present invention can therefore also be represented by StructuralFormula (IX):

R is an organic radical. The term “organic radical”, as it is usedherein, refers to a moiety which comprises primarily hydrogen andcarbon, but can also include small amounts of other non-metallicelements such as sulfur, nitrogen, oxygen and halogens. R, when takentogether with the one or more stabilized S-nitrosyl groups or the one ormore cyclic structures depicted in Strutural Formula (IX), forms a smallorganic molecule, as described above.

n in Structural Formula (IX) is an integer, preferably from one to aboutfive. When n is greater than 1, the stabilized NO-releasing compound hasmore than one stabilized —S-nitrosyl group. Each —S-nitrosyl group in amolecule requires a separate free thiol or separate free alcohol forstabilization.

The compounds of the present invention preferably have a molecularweight less than about 1000 atomic mass units (hereinafter “amu”). Whenthe S-nitrosyl group is stabilized by an alcohol group, the compoundpreferably has a molecular weight greater than about 225 amu and morepreferably greater than about 500 amu. When the S-nitrosyl group isstabilized by a thiol group, the compound preferably has a molecularweight greater than about 375 amu, more preferably greater than about500 amu. When the S-nitrosyl group is stabilized by a thiol group andthe compound has a half-life for NO release of greater than about twohundred hours, the compound preferably has a molecular weight greaterthan about 225 amu.

A polythiol is a small organic molecule which has two or more free thiolgroups. Preferably, a polythiol has between about two and about ten freethiol groups.

A thioalcohol is a small organic molecule which has at least one alcoholgroup and at least one free thiol group. Preferably, a thioalcohol hasone to about five free thiol and one to about five free alcohol groups.

As used herein, the terms “polythiol” and “thioalocohol” do not includepolypeptides or polythiolated polysaccharides and polymers with pendantthiol groups as described in U.S. Pat. No. 5,770,645. The terms“polythiol” and “thioalcohol” also specifically exclude compoundsrepresented by Structural Formulas (I)–(VII).

Suitable nitrosylating agents are disclosed in Feelisch and Stamler,“Donors of Nitrogen Oxides”, Methods in Nitric Oxide Research edited byFeelisch and Stamler, (John Wiley & Sons) (1996), the entire teachingsof which are hereby incorporated into this application by reference.Suitable nitrosylating agents include acidic nitrite, nitrosyl chloride,compounds comprising an S-nitroso group(S-nitroso-N-acetyl-D,L-penicillamine (SNAP), S-nitrosoglutathione(SNOG), N-acetyl-S-nitrosopenicillaminyl-S-nitrosopenicillamine,S-nitrosocysteine, S-nitrosothioglycerol, S-nitrosodithiothreitol andS-nitrosomercaptoethanol), an organic nitrite (e.g. ethyl nitrite,isobutyl nitrite, and amyl nitrite) peroxynitrites, nitrosonium salts(e.g. nitrosyl hydrogen sulfate), oxadiazoles (e.g.4-phenyl-3-furoxancarbonitrile) and the like. The half-life forNO-release of stabilized S-nitrosylated compounds can depend, at leastin part, on the nitrosylating agent used in their preparation. Forexample, the half-life for NO release of stabilized S-nitrosylatedcompounds prepared with SNAP is generally greater than the correspondingcompound prepared with tert-butyl nitrite (Example 2).

To prepare the compounds of the present invention, a polythiol or athioalcohol is reacted with between about 0.5 to about 0.7 equivalentsof nitrosylating agent with respect to each alcohol and thiol.Preferably, nitrosylating agent is added to the polythiol orthioalcohol. For example, to prepare a compound with a stabilizedS-nitrosyl group from 1,2-dithioethane or thioethanol, 1.0 mole ofnitrosylating agent is added to 1.0 mole of 1,2-dithioethane orthioethanol. S-nitroso-N-acetyl-D,L-penicillamine (SNAP) is a preferrednitrosylating agent. Larger amounts of nitrosylating agent with respectto thiol and/or alcohol groups can be used can be used with certainnitrosylating agents such as S-nitroso-N-acetyl-D,L-penicillamine(SNAP).

The nitrosylation reaction can be carried out neat or in a solvent inwhich the polythiol or thioalcohol and the nitrosylating agent aresoluble. Commonly used solvents include dimethyl sulfoxide (DMSO),dimethyl formamide (DMF) and acetonitrile. Reaction temperatures betweenabout 0° C. and about 50° C., preferably ambient temperature, can beused. Concentrations of polythiol or thioalcohol are generally greaterthan about 0.01 M. Specific conditions for nitrosylation with SNAP andtert-butylnitrite (TBN) are provided in Example 1.

The reaction with acidic nitrite as the nitrosylating agent can be, forexample, carried out in an aqueous solution with a nitrite salt, e.g.NaNO₂, KNO₂, LiNO₂ and the like, in the presence of an acid, e.g. HCl,acetic acid, H₃PO₄ and the like, at a temperature from about −20° C. toabout 50° C., preferably at ambient temperature.

The reaction with NOCl as the nitrosylating agent can be carried out,for example, in an aprotic polar solvent such as dimethylformamide ordimethylsulfoxide at a temperature from about −20° C. to about 50° C.,preferably at ambient temperature.

The formation of stabilized S-nitrosyl groups is disfavored when thepolythiol or thioalcohol has substituents, particularly bulkysubstituents, in close proximity to the stabilizing thiol or alcoholgroups. For example, the formation of stabilized S-nitrosyl groups isdisfavored when three substituents, for example three alkyl groups, areattached to the carbon atoms alpha or beta to the thiol. AlthoughApplicants do not wish to be bound by any particular mechanism, it isbelieved that bulky groups in close proximity to a thiol or alcohol cansterically block the interaction between the thiol or alcohol and theS-nitrosyl group. Thus, —Y— and —S— in stabilized S-nitrosyl groups arepreferably bonded to methylene groups.

In a preferred embodiment, the compound is formed by nitrosylating anesterified polyol represented by Structural Formula (X):

R is an organic radical, as described above.

n in Structural Formula (X) is an integer greater than two, preferablyan integer from three to about ten. More preferably, n is an integerfrom three to about eight.

Each X is independently a thiol-bearing aliphatic group or a substitutedthiol-bearing aliphatic group. Preferably, each X is the samethiol-bearing aliphatic group. Examples of suitable thiol-bearingaliphatic groups include —CH₂SH, —CH₂CH₂SH, —CH₂CH₂CH₂SH and—CH₂CH₂CH₂CH₂SH.

The nitrosylation of the esterified polyol is carried out by reactingthe esterified polyol with a nitrosylating agent, as described above,preferably with about 0.5 to about 0.7 equivalents of nitrosylatingagent per free thiol and free alcohol.

Compounds prepared by nitrosylating an esterified polyol represented byStructural Formula (X) have one or more stabilized S-nitrosyl groups.The compound formed by this reaction is represented by StructuralFormula (XI):

R is an organic radical, as described above.

Each X′ is an independently chosen aliphatic group or substitutedaliphatic group. Preferably each X′ is the same and is a C2–C6 alkylgroup, more preferably —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂— or —CH₂CH₂CH₂CH₂—.

p and m are positive integers such that p+m is greater than two.Preferably, p+m is less than or equal to about 10. Even more preferably,p+m are less than or equal to six.

Specific examples of polythiols and thioalcohols which have beennitrosylated to form compounds with stable S-nitrosyl groups are shownin the Table in Example 2. Also shown in the Table are the half-life forNO-release for each S-nitrosylated compound and the nitrosylating agentused to prepare each compound.

Compounds represented by Structural Formula (XI) can form polymers,which can be used to coat medical devices for delivering NO in vivo.These polymers are disclosed in co-pending U.S. patent application Ser.No. 6,232,434, filed on Jun. 23, 1998, the entire teachings of which areincorporated herein by reference.

As used herein, aliphatic groups include straight chained, branched orcyclic C₁–C₈ hydrocarbons which are completely saturated or whichcontain one or more units of unsaturation. Suitable substituents for analiphatic group are those which: 1) are substantially inert with respectto -S-nitrosyl groups, i.e., groups which do not substantially increasethe rate, e.g., double the rate of NO release from NO-releasingmolecules; and 2) do not substantially interfere with the nitrosylationof free thiol groups, i.e. do not substantially decrease the yield ofthe nitrosylation or cause the formation of significant amounts ofby-products. Examples of suitable substituents include halogens, C1–C5straight or branched chain alkyl groups, alcohols, carboxylic acids,amides, thioamides, and the like.

Another embodiment of the present invention is a method of delivering NOto a treatment site in a subject using the novel compositions of thepresent inventions to deliver NO. A “treatment site” includes a site inthe body of a subject in which a desirable therapeutic effect can beachieved by contacting the site with NO. A “subject” refers to a humanor an animal such as a veterinary animal (e.g., dogs, cats and the like)and farm animals (e.g., horses, cows, pigs and the like).

Treatment sites are found, for example, at sites within the body whichdevelop restenosis, injury or thrombosis as a result of trauma caused bycontacting the site with a synthetic material or a medical device. Forexample, restenosis can develop in blood vessels which have undergonecoronary procedures or peripheral procedures with PTCA balloon catheters(e.g. percutaneous transluminal angioplasty). Restenosis is thedevelopment of scar tissue from about three to six months after theprocedure and results in narrowing of the blood vessel. NO reducesrestenosis by inhibiting platelet deposition and smooth muscleproliferation. NO also inhibits thrombosis by inhibiting platelets andcan limit injury by serving as an anti-inflammatory agent.

A site in need of treatment with NO often develops at vascular siteswhich are in contact with a synthetic material or a medical device. Forexample, stents are often inserted into blood vessels to preventrestenosis and re-narrowing of a blood vessel after a procedure such asangioplasty. Platelet aggregation resulting in thrombus formation is acomplication which may result from the insertion of stents. NO is anantiplatelet agent and can consequently be used to lessen the risk ofthrombus formation associated with the use of these medical devices.Other examples of medical devices which contact vascular sites andthereby increase the risk of thrombus formation include sheaths forveins and arteries and GORE-TEX surgical prostheses.

The need for treatment with NO can also develop at non-vascular sites,for example at sites where a useful therapeutic effect can be achievedby reducing an inflammatory response. Examples include the airway, thegastrointestinal tract, bladder, uterine and corpus cavemosum. Thus, thecompositions, methods and devices of the present invention can be usedto treat respiratory disorders, gastrointestinal disorders, urologicaldysfunction, impotence, uterine dysfunction and premature labor. NOdelivery at a treatment site can also result in smooth muscle relaxationto facilitate insertion of a medical device, for example in proceduressuch as bronchoscopy, endoscopy, laparoscopy and cystoscopy. Delivery ofNO can also be used to prevent cerebral vasospasms post hemorrhage andto treat bladder irritability, urethral strictures and biliary spasms.

The need for treatment with NO can also arise external to the body inmedical devices used to treat bodily fluids temporarily removed frombody for treatment, for example blood. Examples include conduit tubeswithin heart lung machines, tubes of a dialysis apparatus and catheters.

The method of delivering NO to a treatment site in a subject containsimplanting a medical device which comprises one or more compounds of thepresent invention at the treatment site. NO can be delivered to bodilyfluids, for example blood, by contacting the bodily fluid with a tube orcatheter comprising one or more compounds of the present invention.Examples of treatment sites in a subject, medical devices suitable forimplementation at the treatment sites and medical devices suitable forcontacting bodily fluids such as blood are described in the paragraphshereinabove.

“Implanting a medical device at a treatment site” refers to bringing themedical device into actual physical contact with the treatment site or,in the alternative, bringing the medical device into close enoughproximity to the treatment site so that NO released from the medicaldevice comes into physical contact with the treatment site. A bodilyfluid is contacted with a medical device, e.g., a tube or cather, whichcomprises one or more compounds of the present invention when, forexample, the bodily fluid is temporarily removed from the body fortreatment by the medical device, and the coating is an interface betweenthe bodily fluid and the medical device. Examples include the removal ofblood for dialysis or by heart lung machines.

An article, for example a medical device such as a stent, tube orcatheter, can be coated with one or more compounds of the presentinvention. A mixture is formed by combining a solution comprising adithiol or thioalcohol with an article insoluble in the solution. Themixture is then combined with a nitrosylating agent under conditionssuitable for nitrosylating free thiol groups, resulting in formation ofa stabilized NO-releasing compound. When the stabilized NO-releasingcompound is insoluble in solution, the NO-releasing compoundprecipitates from the solution and coats the article. When thestabilized NO-releasing compound is soluble in the solution or when thenitrosylation reaction is carried out in a polar aprotic solvent such asdimethylformamide (DMF) or dimethylsulfoxide (DMSO), the article can bedipped into or sprayed with the reaction mixture and then dried in vacuoor under a stream of an inert gas such as nitrogen or argon, therebycoating the article. Suitable nitrosylating agents include SNAP,tert-butyl nitrite, acidified nitrite, S-nitrosothiols, organic nitrite,nitrosyl chloride, oxadiazoles, nitroprusside and other metal nitrosylcomplexes, peroxynitrites, nitrosonium salts (e.g. nitrosylhydrogensulfate) and the like.

It is to be understood that other methods of applying coatings todevices, including methods known in the art, can be used to coatarticles with the compounds of the present invention.

An article incorporates an NO-releasing compound of the presentinvention when the compound is “entrapped” within the molecularframework of a material which is part of the article. For example, manymedical devices include certain polymers. An NO-releasing compound canbe incorporated into these polymers by carrying out the polymerizationreaction through which these polymers are formed in the presence of anNO-releasing compound. The NO-releasing compound is thereby entrapped inthe molecular framework of the resulting polymer product, which can thenbe used to prepare the medical device.

An article also incorporates an NO-releasing compound when theNO-releasing compound is chemically bonded to a material which is partof the article.

It is to be understood that other methods of incorporating compoundsinto compositions are known in the art and can be used to incorporatethe compounds of the present invention into the materials used toproduce medical devices.

The NO-releasing compounds of the present invention are bacteriostatic(Example 3). Thus, these compounds can be used to inhibit the growth ofbacteria on surfaces, for example, the surfaces of medical devices ormedical furniture prior to use. “Inhibiting the growth of bacteria”refers to a statistically significant lower bacteria count on a surfaceafter application of the compound compared with a similar surface whichhas not been treated with the compound. The NO-releasing compound isapplied to a surface in need of sterilization by, for example,dissolving the compound in a non-toxic solvent at concentrations betweenabout 0.01 M and 5.0 M. The solution is then applied to the surface byspraying, wiping or pouring the resulting solution onto said surface. Aquantity of solution sufficient to cover the surface is generally used.The solution is allowed to remain in contact with the surface for aslong as inhibition of bacteria growth is required. The solution and anyresidues are removed, for example, by wiping or washing with a solventsuitable for dissolving the NO-releasing compound and any decompositionproducts.

The invention is further illustrated by the following examples, whichare not intended to be limiting in any way.

EXEMPLIFICATION

All precursor thiols were obtained from Sigma-Aldrich Chemical Co. andwere used without further purification. Tertiary-butyl nitrite (TBN,96%) and N-acetyl-D,L-penicillamine were purchased from Aldrich ChemicalCo. and were used without further purification. Dimethylsulfoxide (DMSO)was purchased from Mallinckrodt, passed through a CHELEX 100 column andbubbled for thirty minutes with argon prior to use. Dimethylformamide(DMF) was purchased from VWR Scientific.

S-Nitrosyl-N-acetyl-D,L-penicillamine (SNAP) was prepared by mixing a0.2 M solution of N-acetyl-D,L-penicillamine in 1:1 methanol/1 N HClwith an equimolar amount of sodium nitrite in water. SNAP precipitatedout of solution as a green solid, which was filtered and washed withwater prior to use.

EXAMPLE 1 Preparation of Stabilized S-Nitrosylated Compounds

S-nitrosylated compounds were prepared by adding TBN to a polythiol orthioalcohol. Alternatively, S-nitrosylated compounds were prepared byadding a polythiol or thioalcohol to a solution of SNAP in DMF (0.12 Mis a representative concentration). All reactions were carried out atroom temperature. 0.5 equivalents of nitrosylating agent per thiol group(or per alcohol and thiol group) were used. All reactions were carriedout under argon in the dark. A rapid color change to red was observedafter addition of the nitrosylating agent. Stirring was continued forapproximately another two to five minutes after the color change.

S-nitrosylated compounds were characterized by ¹⁵N NMR and by theirUV/visible absorbance spectra. The ¹⁵N NMR spectrum of stabilizedS-nitrosylated compounds showed a singlet at about 424 ppm relative toHNO₃. In contrast, the ¹⁵N NMR spectrum of S-nitrosylated tertiary butylthiol prepared according to the procedure described above gave a singletat 480 ppm relative to HNO₃. The UV/visible absorbance spectrum ofstabilized S-nitrosylated compounds gave an absorbance maximum betweenabout 540 and 555 nanometers.

EXAMPLE 2 Half-Lives for NO Release From Stabilized S-NitrosylatedCompounds

The half-lives for NO release from the stabilized S-nitrosylatedcompounds of the present invention were determined by monitoring thedecrease in intensity of the absorbance maximum between 540 and 555nanometers over time. When the nitrosylation reaction was performedneat, the uv/visible absorbance spectrum was obtained by adding severaldrops of the reaction mixture to a cuvette containing DMSO. When thenitrosylation was carried out in solution, the absorbance spectrum wasobtained directly from the reaction mixture. Data for these plots wereobtained by performing at least three absorbance scans to create alinear plot from which half-lives were determined. Kinetics runs wereperformed in the dark under an argon atmosphere. A Hewlett-Packard 8452ADiode Array Spectrophotometer in conjunction with HP8953 IA MS-DOSUV/VIS Operating Software was utilized to obtain kinetic data for use inhalf-life determinations.

The Table shows the half-lives for NO release of a number of stabilizedS-nitrosylated polythiols and thioalcohols prepared according to theprocedures described in Example 1. The Table indicates whether thehalf-life determination was for an S-nitrosylated compound prepared withSNAP or TBN. Only approximate values could be determined when thehalf-life was greater than one thousand hours.

The Table also shows that the half-live for NO release forS-nitrosylated hexane thiol prepared with SNAP according to theprocedure described above is 280 hours and that the half-life for NOrelease of 1—S-nitrosyl hexane 6-thiol prepared with SNAP is about 1800hours. This result demonstrates the stabilizing effect of the free thiolgroup.

As can be seen from the Table, longer half-lives are generally obtainedwhen SNAP was used as the nitrosylating agent. S-Nitrosylated compoundsobtained from SNAP in the Table generally have half-lives greater thantwo hundred hours.

TABLE Half-Life For NO-Release in Hours: Compound TBN¹ SNAP²

104  225

87  570

84  618

108

126  440

167  170

196 1500³

105  228

386 2200³

33  150

48.8 1300³

207.2 1800³

 188

 280 ¹Half-life for NO release when the compound is nitrosylated withTBN ²Half-life for NO release when the compound is nitrosylated withSNAP ³Approximate half-lives

EXAMPLE 3 Nitrosylated 3-Mercapto-1,2-Propanediol is Bacteriostatic

Nitrosated 3-mercapto-1,2-propanediol was prepared with TBN bed in theExample 1. The nitrosylated compound was dissolved in 10 mL of deionizedwater (0.16 M) that had been passed through a CHELEX 100 column andbubbled with argon for 30 minutes. The resulting solution was red.

E. coli cells (pTC 190) were plated onto the culture plates and grownovernight at 40° C. The E. coli cells contained a plasmid encoding forampicillin resistance. The solution was then sprayed onto a cultureplate (LB/Amp/Glucose). After 24 hours, this plate showed the growth ofa few colonies, all localized on one side of the plate.

A growth plate coated only with E. coli cells displayed a continuouslawn of growth after 24 hours. This plate was then sprayed with thenitrosylated compound solution at the 24^(th) hour. After another 24hours the plate contained a lawn of colonies that was approximately asdense as prior to application of the S-nitrosylated compound.

A control plate that was not coated with the nitrosylated compoundsolution nor plated with E. coli cells showed no bacterial growth. Asecond control plate that was not plated with E. coli cells but coatedwith the nitrosylated compound solution showed no bacterial colonygrowth.

These results show that nitrosylated 3-mercapto-1,2-propanediol isbacteriostatic.

EXAMPLE 4 Reaction of 1,6-Hexanedithiol and 1-Hexanethiol with SNAP

1,6-Hexanedithiol (25 uL, 24.6 mg, 0.164 mmol) was dissolved in 6.0 mLof DMSO in 11 separate 10-mL test tubes. S-Nitroso-N-acetylpenicillamine(SNAP) was added in incremental stoichiometric amounts to each test tubeas follows:

mg SNAP equivalents SNAP 9.0 0.25 18.0 0.5 27.0 0.75 36.0 1.0 45.0 1.2554.0 1.5 63.0 1.75 72.0 2.0 108.0 3.0 144.0 4.0 180.0 5.0The samples were stored under ambient atmosphere in the dark. Absorbancevalues were taken at time intervals in a 1-cm path length quartz cell atwavelengths of 520, 554, and 594 nm. While the absorbance readings werebeing taken, the samples were under ambient light for approximately 1hour.1-Hexanethiol (25 uL, 21.0 mg, 0.177 mmol) was dissolved in 6.0 mL ofDMSO in 9 separate 10-mL test tubes. S-Nitroso-N-acetylpenicillamine(SNAP) was added in incremental stoichiometric amounts to each test tubeas follows:

mg SNAP equivalents SNAP 9.8 0.25 19.5 0.5 29.3 0.75 39.0 1.0 48.8 1.2558.5 1.5 78.0 2.0 117.0 3.0 156.1 4.0

The samples were stored under ambient atmosphere in the dark. Absorbancevalues were taken at time intervals in a 1-cm path length quartz cell atwavelengths of 520, 554, and 594 nm. While the absorbance readings werebeing taken, the samples were under ambient light for approximately 1 h.

In both reactions, a red color corresponding to an absorbance at 554 nmappeared almost immediately after the reagents are mixed. The red colorpeaked at the same maximum value about three hours after mixing for both1-hexanethiol and 1,6-hexanedithiol when 0.5 equivalents or more of SNAPare used. This result is consistent with the formation of a stabilizedS-nitrosylated compound, i.e., the nitrosylation of only one of thethiol groups in 1,6-hexanedithiol, even when more than 0.5 equivalentsof SNAP are present.

When the experiment was repeated with TBN or acidic nitrite in place ofSNAP, the absorbance peak at 554 nm for the reaction with1,6-hexanedithiol increased as the amount of nitrosylating usedincreased, up to 1.0 equivalent of nitrosylating agent per thiol group.This result is consistent with the formation of stabilizedS-nitrosylated when 0.5 equivalents of TBN or acidic nitrite were used,and with nitrosylation of the second thiols group when more than 0.5equivalents of TBN or acidic nitrite were used.

EQUIVALENTS

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims. Those skilled in the artwill recognize or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described specifically herein. Such equivalents are intendedto be encompassed in the scope of the claims.

1. A compound containing at least one S-nitrosyl group, at least onefree thiol group or free alcohol group, optionally one or morefunctional groups selected from the group consisting of alcohols,thiols, amides, thioamides, aldehydes, ketones, halogens, double bonds,triple bonds and aryl groups wherein the compound has a molecular weightof at least 225 atomic mass units, wherein the S-nitrosyl group isstabilized by the free alcohol group or the free thiol group and whereinthe compound has a half-life for NO-release that is at least two timesas great as the corresponding compound without the free thiol group orfree alcohol group, with the proviso that the polythiol or thioalcoholis not a polypeptide, an unsubstituted straight chained or brancheddithiolated alkane, an unsubstituted straight chained or branchedthioalkanol, a polythiolated polysaccharide, or a polymer with pendantthiol groups and is not represented by one of the following structuralformulas:

wherein each Ra is —H or methyl and is independently chosen.
 2. Thecompound of claim 1, wherein the compound is prepared by reacting apolythiol or a thioalcohol with a nitrosylating agent.
 3. The compoundof claim 2, wherein 0.5 to 0.7 equivalents of nitrosylating agent areused per free thiol and per free alcohol in the polythiol orthioalcohol.
 4. The compound of claim 3, wherein the nitrosylating agentis reacted with a thioalcohol.
 5. The compound of claim 4, wherein thecompound has a molecular weight less than 1000 atomic mass units.
 6. Thecompound of claim 5, wherein the alcohol and thiol are both primary. 7.The compound of claim 3, wherein the nitrosylating agent is reacted witha polythiol and the compound has a molecular weight greater than 375atomic mass units.
 8. The compound of claim 7, wherein the compound hasa molecular weight less than 1000 atomic mass units.
 9. The compound ofclaim 8, wherein both thiols are primary.
 10. The compound of claim 1,wherein the nitrosylating agent is S-nitroso-N-acetyl-D,L-penicillamine.11. A method of preparing a compound containing a stabilized S-nitrosylgroup, a free thiol group or free alcohol group and optionally one ormore functional groups selected from the group consisting of alcohols,thiols, amides, thioamides, aldehydes, ketones, halogens, doublebonds,triple bonds and aryl groups, wherein the compound has a molecularweight of at least 225 atomic mass units, wherein the S-nitrosyl groupis stabilized by the free alcohol group or the free thiol group andwherein the compound has a half-life for NO-release that is at least twotimes as great as the corresponding compound without the free thiolgroup or free alcohol group, said method comprising the step of reactinga polythiol or a thioalcohol with a nitrosylating agent, with theproviso that the polythiol or thioalcohol is not a polypeptide, anunsubstituted straight chained or branched dithiolated alkane, anunsubstituted straight chained or branched thioalkanol, a polythiolatedpolysaccharide, or a polymer with pendant thiol groups and is notrepresented by one of the following structural formulas:

wherein each Ra is —H or methyl and is independently chosen.
 12. Themethod of claim 11, wherein 0.5 to 0.7 equivalents of nitrosylatingagent per free thiol and per free alcohol in the polythiol orthioalcohol is used.
 13. The method of claim 12, wherein thenitrosylating agent is reacted with a thioalcohol.
 14. The method ofclaim 13, wherein the compound has a molecular weight less than 1000atomic mass units.
 15. The method of claim 14, wherein the alcohol andthiol are both primary.
 16. The method of claim 12, wherein thenitrosylating agent is reacted with a polythiol and the compound has amolecular weight greater than 375 atomic mass units.
 17. The method ofclaim 16, wherein the compound has a molecular weight less than 1000atomic mass units.
 18. The method of claim 17, wherein both thiols areprimary.
 19. The method of claim 11, wherein the nitrosylating agent isS-nitroso-N-acetyl-D,L-penicillamine.